Effective mechanisms to combat stress are vital to all living organisms. We study the fundamental design principles of the signal transduction cascades that sense temperature stress in E. coli. Interestingly, cells compartmentalize their stress responses. The cytoplasmic heat shock response (HSR), controlled by sigma32, regulates expression of the universally conserved heat shock proteins (hsps). The envelope stress response (ESR), controlled by sigmaE, regulates expression of proteins required to maintain envelope integrity. The proposed studies will define the cellular inputs that negatively regulate the HSR and define the protease cascade that regulates the ESR by: 1. Determining how chaperone-mediated inputs to the Hsr are partitioned between GroEL/S and DnaK/J and the features of sigma32 making it amenable to chaperone mediated inactivation. 2. Testing whether FtsH protease plays a regulatory role in the HSR by determining when changes in substrate occupancy of FtsH alter sigma32 activity. 3. Finding and characterizing the factor necessary to obtain rapid degradation of sigma32 by FtsH protease in vitro. 4. Determining how RseB facilitates sequential degradation of RseA, the sigmaE-specific antisigma by preventing inappropriate cleavage of intact RseA by YaeL, the second protease in the pathway. 5. Testing whether a DegS-YaeL machine mediates concerted degradation of RseA. 6. Identifying the proteases that complete RseA degradation and demonstrating their function in vitro. 7. Identifying additional regulatory inputs to the ESR to understand the broader role of this response and whether any signals work at different steps in the protease cascade. Given the universality of these responses and the increasing evidence that many organisms choose similar general solutions to deal with stress, our studies of these signal transduction cascades have broad applicability to the understanding of the successful design of such circuits in all organisms. In addition, the proteases carrying out these responses, which are most easily studied in E. coli, are poorly understood, but widely distributed, often mediating stress responses in organisms ranging from microbial pathogens to man. Finally, studies on the heat shock response has direct medical relevance as T cells reactive to heat shock proteins may provide a first line of defense against infection, heat shock proteins are often up-regulated in cancer and the increasing variety of protein folding diseases makes it imperative to understand how the protein folding state of the cell is maintained.